This PDF file contains the front matter associated with SPIE Proceedings Volume 10497, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

Macrophages are essential for the regeneration of skeletal muscle after injury. It has been demonstrated that depletion of macrophages results in delay of necrotic fiber phagocytosis and decreased size of regenerated myofibers. In this work, we developed a multi-modal two-photon microscope system for in vivo study of macrophage activities in the regenerative and fibrotic healing process of injured skeletal muscles. The system is capable to image the muscles based on the second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) signals simultaneously. The dynamic activities of macrophages and muscle satellite cells are recorded in different time windows post the muscle injury. Moreover, we found that infiltrating macrophages emitted strong autofluorescence in the injured skeletal muscle of mouse model, which has not been reported previously. The macrophage autofluorescence was characterized in both spectral and temporal domains. The information extracted from the autofluorescence signals may facilitate the understanding on the formation mechanisms and possible applications in biological research related to skeletal muscle regeneration.

Fluorescence molecular imaging (FMI) has been widely used in many medical fields with small molecule indocyanine green (ICG). However, low signal-background ratio and limited specificity to tumor remain big challenges for FMI. In this study, a novel excitation strategy is proposed on the basis of clinical approved ICG and ¹⁸F-FDG. A series of in vitro experiments are designed to reveal the mechanism and results show obvious decreasing of ICG fluorescence intensity with the increasing distance to excitation source. Meanwhile, the ICG fluorescence intensity is proportional to the activity of radiopharmaceutical. Results from different respects illustrate the promising of this proposed excitation strategy.

The majority of microscopic and endoscopic technologies utilize white light illumination. For a number of applications, hyper-spectral imaging can be shown to have significant improvements over standard white-light imaging techniques. This is true for both microscopy and in vivo imaging. However, hyperspectral imaging methods have suffered from slow application times. Often, minutes are required to gather a full imaging stack. Here we will describe and evaluate a novel excitation-scanning hyperspectral imaging system and discuss some applications. We have developed and are optimizing a novel approach called excitation-scanning hyperspectral imaging that provides an order of magnitude increased signal strength. This excitation scanning technique has enabled us to produce a microscopy system capable of high speed hyperspectral imaging with the potential for live video acquisition. The excitation-scanning hyperspectral imaging technology we developed may impact a range of applications. The current design uses digital strobing to illuminate at 16 wavelengths with millisecond image acquisition time. Analog intensity control enables a fully customizable excitation profile. A significant advantage of excitation-scanning hyperspectral imaging is can identify multiple targets simultaneously in real time. Finally, we are exploring utilizing this technology for a variety of applications ranging from measuring cAMP distribution in three dimensions within a cell to electrophysiology.

In most deaths caused by explosions, the fragment of explosive remained on the victim's body can provide valuable clues to forensic investigation. However, the examination of the skin and appendages at the scene of explosion, which may reveal clue to the identity of an individual, has not been extensively studied. Compared with visual appearance of the epidermis surface that affected by various wounds, skin adnexa embedded in the dermis has a more stable morphology as an inner biometric. Hair follicles are formed when a fetus is 5 months old and distributed fairly evenly throughout the body, with the exception of hairless palms and soles. Therefore, we focus on the distribution of hair follicles in order to infer information of age, gender, and race/ethnicity. Optical coherence tomography (OCT) is a novel forensic imaging method, which achieves non-destructive, high-resolution and most importantly cross-sectional imaging. In this study, we design and develop a custom-built spectral-domain three-dimensional (3D) OCT system with a portable handheld probe to detect and reconstruct the hair follicles in the facial skin. We test our system on the forehead and preauricular skin of 2 adult volunteers and demonstrate the high quality visualization of hair follicles beneath the epidermis. The diameter, orientation, density and shape of hair follicles can be extracted from the 3D volume data. The preliminary analysis suggests that these parameters vary from different part of body and have individual difference. Eventually, we believe 3D OCT is promising tool for the examination of hair follicles for forensic purpose.

Objective: This study utilizes fluorescence cryoimaging to quantitatively assess the effect of a high dose of irradiation on rat renal metabolism through redox state. Introduction: Exposure to high doses of irradiation could lead to death, in part, due to renal dysfunction. The kidney is one of the most sensitive organs that exhibit delayed injuries in survivors of acute radiation syndrome. In this study, optical cryoimaging was utilized to examine the potential for renal mitochondrial dysfunction after partial-body irradiation (PBI) and the mitigating effect of lisinopril-treatment, an angiotensin converting enzyme inhibitor that is FDA-approved for other indications. Materials and methods: Rats were exposed to a single dose of 13 Gy leg-out partial body irradiation (PBI, by X-rays). Rats (n = 5/group) received no further treatment, or lisinopril started one week after irradiation and continued at 24 mg/m² /day. The non-irradiated siblings were used as controls. After 150 days, the rats were sacrificed, and their kidneys harvested and snap frozen in liquid nitrogen for later cryoimaging. The 3D images of metabolic indices (NADH and FAD) were captured, and the redox ratio i.e. NADH/FAD was calculated. The mitochondrial redox state of three groups of rat kidneys were quantified by calculating the volumetric mean of redox ratio images (RR). Results: 3D cryoimaging revealed that in PBI only kidneys, the metabolic marker (RR) decreased significantly by 78% compared to non-irradiated controls. Treatment with lisinopril significantly improved the RR by 93% in groups exposed to PBI. Conclusion: This study aimed at quantifying the level of the mitochondrial redox state of irradiated rat kidneys compared to non-irradiated kidneys (controls) and the efficacy of lisinopril to preserve kidney metabolism after irradiation. PBI oxidized the metabolic state of kidneys and lisinopril mitigated the radiation-induced injury on renal mitochondria.

High-throughput in vitro screening of highly physiological three-dimensional cell cultures (3D-HTS) is rapidly gaining importance in preclinical studies, to study the effect of the microenvironment in tumor development, and to evaluate the efficacy of new anticancer drugs. Furthermore, it could also be envisioned the use of 3D-HTS systems in personalized anti-cancer treatment planning, based on tumor organoids or spheroids grown from tumor biopsies or isolated tumor circulating cells. Most commercial, multi-well plate based 3D-HTS systems are large, expensive, and are based on the use of multi-well plates that hardly provide a physiological environment and require the use of large amounts of biological material and reagents. In this paper we present a novel, miniaturized inverted microscope (hereinafter miniscospe), made up of low-cost, mass producible parts, that can be used to monitor the growth of living tumor cell spheroids within customized three-dimensional microfluidic platforms. Our 3D-HTS miniscope combines phase contrast imaging based on oblique back illumination technique with traditional widefield epi-fluorescence imaging, implemented using miniaturized electro-optical parts and gradient-index refraction lenses. This small (3x6x2cm), lightweight device can effectively image overtime the growth of (>200) tumor spheroids in a controlled and reproducible environment. Our miniscope can be used to acquire time-lapse images of cellular living spheroids over the course of several hours and captures their growth before and after drug treatment, to evaluate the effectiveness of the drug.

We introduce a label-free technique based on lens-free microscopy to perform cell counting and cell viability assay. Without the use of any labelling, the discrimination between alive and dead cells is obtained by analyzing the cells on the basis of their holographic signature. We have assessed this novel technique by comparing the obtained results in terms of viability and cell counting against automatic optical counting and trypan blue staining as reference methods. The lensfree measurements agree very well with the reference techniques up to ~30.10⁶ cells/ml. We found a coefficient of determination R² of 0.99 and a slope of 1.01 for the viability measurements (N=84 CHO cell samples).

Monitoring of variations in morphological characteristics of cultured HeLa cells after photodynamic treatment with Radachlorin photosensitizer is performed by means of digital holographic microscopy. The observed dose-dependent post-treatment variations of phase shift evidence threshold effect of photodynamic treatment and allow for distinguishing between necrotic or apoptotic pathways of cell death. Results obtained by holographic microscopy were confirmed by means of far-field optical microscopy and confocal fluorescence microscopy with commonly used test assays.

A precise control and a reliable monitoring tool for the adhesion properties of a cell are very important in atherosclerosis studies. If endothelial cells in contact with the intracellular membrane are not attached securely, low-density lipoprotein (LDL) particles can enter into the inner membrane. It is therefore necessary to measure conditions under which endothelial cell detachment occurs. When a cell is attached to a metal thin film, the lifetime of a fluorescence probe attached to the membrane of the cell is reduced by the metal-induced energy transfer (MIET). Fluorescence lifetime imaging microscopy (FLIM) is used to monitor the attachment condition of a cell to a metal surface using FRET. However, this requires high numerical aperture (NA) objective lens because axial confocal resolution must be smaller than the cell thickness. This requirement limits the field of view of the measurement specimen. In this study we provides a new method which can measure adhesion properties of endothelial cells even with a low NA objective lens by resolving two lifetime components in FLIM.

Otical imaging techniques based on time correlated single photon counting (TCSPC) has found wide applications in medicine and biology. Non-invasive and information-rich fluorescence lifetime imaging microscopy (FLIM) is successfully used for monitoring fluorescent intrinsic metabolic coenzymes as NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) and FAD+ (flavin adenine dinucleotide) in living cells and tissues. The ratio between proteinbound and free coenzymes gives an information about the balance between oxidative phosphorylation and glycolysis in the cells. The changes of the ratio reflects major cellular disorders. A correlation exists between metabolic activity, redox ratio and fluorescence lifetime during stem cell differentiation, neurodegenerative diseases, and carcinogenesis.

A multichannel FLIM detection system was designed for monitoring the redox state of NAD(P)H and FAD+ and other intrinsic fluorophores as protoporphyrin IX. In addition, the unique upgrade is useful to perform FLIM and PLIM (phosphorescence lifetime imaging microscopy) simultaneously. PLIM is a promising method to investigate oxygen sensing in biomedical samples. In detail, the oxygen-dependent quenching of phosphorescence of some compounds as transition metal complexes enables measuring of oxygen partial pressure (pO₂). Using a two-channel FLIM/PLIM system we monitored intrinsic pO₂ by PLIM simultaneously with NAD(P)H by FLIM providing complex metabolic and redox imaging of living cells.

Physico-chemical properties of oxygen sensitive probes define certain parameters including their localisation. We present results of some ruthenium based complexes including those specifically bound to mitochondria.

The present standard of blood cell analysis is an invasive procedure requiring the extraction of patient’s blood, followed by ex-vivo analysis using a flow cytometer or a hemocytometer. We are developing a noninvasive optical technique that alleviates the need for blood extraction. For in-vivo blood analysis we need a high speed, high resolution and high contrast label-free imaging technique. In this proceeding report, we reported a label-free method based on differential epi-detection of forward scattered light, a method inspired by Jerome Mertz's oblique back-illumination microscopy (OBM) (Ford et al, Nat. Meth. 9(12) 2012). The differential epi-detection of forward light gives phase contrast image at diffraction-limited resolution. Unlike reflection confocal microscopy (RCM), which detects only sharp refractive index variation and suffers from speckle noise, this technique is suitable for detection of subtle variation of refractive index in biological tissue and it provides the shape and the size of cells. A custom built high speed electronic detection circuit board produces a real-time differential signal which yields image contrast based on phase gradient in the sample. We recorded blood flow in-vivo at 17.2k lines per second in line scan mode, or 30 frames per second (full frame), or 120 frame per second (quarter frame) in frame scan mode. The image contrast and speed of line scan data recording show the potential of the system for noninvasive blood cell analysis.

Adipose-derived stem cells (ADSC) based therapies have the potential to treat cartilage disorders such as osteoarthritis in both animals and humans. The current opinion points to secretions from stem cells in the proximity of the diseased joints as key agents in these new regenerative treatments. In this work, biological insights are derived from native fluorescence by using hyperspectral imaging technique. This approach allows to noninvasively monitor the relative levels of individual biochemicals in the tissue, at a cellular level. In this work, we used hyperspectral imaging and unsupervised unmixing to characterize the effects of specific cytokine treatments on different cartilage layers. These layers are composed of various types of collagen, elastin as well as chondrocytes. Strong autofluorescence of the cartilage chip especially from collagen type II presents a significant challenge for the hyperspectral unmixing analysis because the background signal from the collagen tends to dominate the autofluorescence from the chondrocytes. The latter is used for therapy monitoring, and it allows to compare the merits of specific regenerative treatments of cartilage. The unsupervised hyperspectral unmixing approach developed in this work, Robust Dependent Component Analysis (RoDECA) provides a robust and detailed biochemical information from the examined cells and tissues, with a proper account of intrinsic cellular heterogeneity. With appropriate hardware adjustment and software modification in this cartilage chip model study, it was possible to analyze the biochemical effects of the stem cell based treatments. Most importantly, collagen I and collagen II have been distinguished for the first time in a label- free manner, which gives a new way of observing the regenerative treatments of the articular cartilage. This method can be extended in the future to the analysis of optically thick tissue.

Cell fate mapping and lineage tracing are significant ways to understanding the developmental origins of biological tissues. It requires labeling individual cells and tracing the development of their progeny. We develop an infrared laser-evoked gene operator heat-shock microscope system to achieve single-cell labeling in zebrafish. With a fluorescent thermometry technique, we measure the temperature increase in zebrafish tissues induced by infrared laser and identify the optimal heat shock conditions for single-cell gene induction in different types of zebrafish cells. We use this technique to study the fate mapping of T lymphocytes and discover the distinct waves of lymphopoiesis during the zebrafish development.

Focused femtosecond-laser pulses can create tiny transient holes in cell membranes which temporarily allows foreign genetic material from the outside to reach the cell interior. With suitable laser parameters this all optical “optoporation” allows highly efficient laser-assisted cell transfection by reprogramming the cell´s genetic code with very high cell survival rates. Furthermore, the use of viruses or nanoparticle as carriers which may cause serious side effects can be completely omitted. However, the cell positions need to be precisely determined to allow individual focusing of the laser radiation onto the cell membranes which is for large cell numbers quite elaborate and time consuming. We addressed these issues and present optical microscope add-ons for fast and almost hands-off laserassisted poration of cell membranes with automated determination of the positions of adherent cells in a culture dish or targeting continuously flowing cells in suspension.

Engineered tissues offer great promise as engrafted therapies and in vitro models, but these tissues require a vascular network to retain viability at large scales. Significant efforts are focused on optimizing these in vitro vascular constructs, yet current evaluation methods require fixation and immunostaining. These destructive evaluation methods alter vascular network morphology, and cannot non-invasively monitor vascular assembly over time. Here, we demonstrate that autofluorescence multiphoton microscopy (MPM) can quantitatively assess the morphology of living 3D vascular networks without fixation, labels, or dyes. Autofluorescence MPM was used to non-invasively monitor the effect of culture conditions on 3D vascular network formation. Human embryonic stem (ES) cell-derived endothelial cells and primary human pericytes cultured in polyethylene glycol (PEG) hydrogels self-assembled into 3D vascular networks. Autofluorescence MPM of the metabolic co-enzyme NAD(P)H (excitation/emission wavelengths of 750 nm/400-460 nm) was used to quantify morphological parameters at day 6 of culture. Specifically, vessel diameter, vascular density, branch point density, and integration of endothelial cells into the network were quantified. Dynamic culture conditions (flow at 1μL/sec) led to vascular networks with higher mean vessel diameter compared to static culture (p<0.05). Standard immunohistochemistry found that vascular networks were positive for markers of endothelial cells, pericytes, and tight junctions. Scanning electron micrographs confirmed vessel lumen formation with pericytes wrapped around vessels. Dye transit of FITC-dextran through the network confirmed leaky endothelial barrier function. Our results demonstrate that autofluorescence MPM can non-invasively evaluate in vitro 3D vascular networks, and could be used for quality control of engineered tissues.

A smartphone mobile medical application is presented, that provides analysis of the health of skin on the face using a smartphone image and cloud-based image processing techniques. The mobile application employs the use of the camera to capture a front face image of a subject, after which the captured image is spatially calibrated based on fiducial points such as position of the iris of the eye. A facial recognition algorithm is used to identify features of the human face image, to normalize the image, and to define facial regions of interest (ROI) for acne assessment. We identify acne lesions and classify them into two categories: those that are papules and those that are pustules.

Automated facial acne assessment was validated by performing tests on images of 60 digital human models and 10 real human face images. The application was able to identify 92% of acne lesions within five facial ROIs. The classification accuracy for separating papules from pustules was 98%.

Combined with in-app documentation of treatment, lifestyle factors, and automated facial acne assessment, the app can be used in both cosmetic and clinical dermatology. It allows users to quantitatively self-measure acne severity and treatment efficacy on an ongoing basis to help them manage their chronic facial acne.

Abstract: Flow Cytometry Standard, FCS, and Digital Imaging and Communications in Medicine standard, DICOM, are based on extensive, superb domain knowledge, However, they are isolated systems, do not take advantage of data structures, require special programs to read and write the data, lack the capability to interoperate or work with other standards and FCS lacks many of the datatypes necessary for clinical laboratory data. The large overlap between imaging and flow cytometry provides strong evidence that both modalities should be covered by the same standard.

Method: The XML Schema Definition Language, XSD 1.1 was used to translate FCS and/or DICOM objects. A MIFlowCyt file was tested with published values.

Results: Previously, a significant part of an XML standard based upon a combination of FCS and DICOM has been implemented and validated with MIFlowCyt data. Strongly typed translations of FCS keywords have been constructed in XML. These keywords contain links to their DICOM and FCS equivalents.

For a precise characterization of time-domain fluorescence lifetime imaging microscopy (FLIM) datasets, an initial processing step is needed to identify the fluorescent impulse response (FIR) at each spatial point in the sample. Hence departing from the measured fluorescent decays, the FIRs are estimated by using the instrument response function (IRF), and this processing step is known as deconvolution. However, the deconvolution methodology requires an initial measurement of the IRF and a corresponding synchronization step with the fluorescent decays. In this context, we propose a blind deconvolution strategy that estimates jointly the FIRs and the IRF in the dataset. For this purpose, each FIR is modeled by a multi-exponential structure. In this way, the FIRs are characterized by the scaling coefficients and time constants of the exponential terms. Meanwhile, there is no explicit model or pre-defined shape for the IRF. Overall estimation process is achieved by an alternated least squares methodology between the FIRs and IRF. First, if the IRF is fixed, a nonlinear least squares framework computes the FIRs parameters at each spatial point of the sample. Meanwhile, once the FIRs are fixed, the samples of the IRF are estimated by a non-negative least squares methodology and using the whole dataset. These alternated optimization steps are performed until a convergence criterion is fulfilled. The proposed blind deconvolution strategy was validated by synthetic datasets and in vivo FLIM oral mucosa measurements. In these tests, our proposal shows good characterizations of the FIRs and the IRFs in the FLIM datasets.

Islet transplantation has been used to treat diabetes, but is faced with various challenges such as the unavailability of donor organs and the requirement for immunosuppression. Islet encapsulation is a promising solution to some of these problems, helping to achieve insulin independence over longer time. Currently, the standard method to evaluate the encapsulation quality is based on manual microscopic screening of the sample, which is extremely low-throughput and laborious, due to the limited field-of-view (FOV) of standard optical microscopes. Here we report a high-throughput islet encapsulation quality screening system based on lens-free on-chip imaging, which can image/analyze over ~8,000 microcapsules in a single shot. The system utilizes a large format CMOS image sensor that has 60 megapixels, providing a sample FOV of 18.15cm2, which is >100-fold larger than the FOV of a lens-based optical microscope. The encapsulated islets are loaded into a custom-made chamber, which is placed onto the image sensor chip with <2mm gap between the sample and sensor planes. A blue LED provides illumination of the sample, casting in-line holograms of the islets onto the image sensor, which is then analyzed by a custom-written image reconstruction and processing software. The total count of the microcapsules, their size, intactness and whether they contain an islet or not, are analyzed, with the results provided to the user. Being high-throughput, low-cost and simple, this platform can be used for researchers to develop encapsulation protocols as well as a quality control tool before the actual transplantation into patients.

Design of portable microfluidic flow/image cytometry devices for measurements in the field (e.g. initial medical diagnostics) requires careful design in terms of power requirements and weight to allow for realistic portability. True portability with high-throughput microfluidic systems also requires sampling systems without the need for sheath hydrodynamic focusing both to avoid the need for sheath fluid and to enable higher volumes of actual sample, rather than sheath/sample combinations. Weight/power requirements dictate use of super-bright LEDs with top-hat excitation beam architectures and very small silicon photodiodes or nanophotonic sensors that can both be powered by small batteries. Signal-to-noise characteristics can be greatly improved by appropriately pulsing the LED excitation sources and sampling and subtracting noise in between excitation pulses.

Microfluidic cytometry also requires judicious use of small sample volumes and appropriate statistical sampling by microfluidic cytometry or imaging for adequate statistical significance to permit real-time (typically in less than 15 minutes) initial medical decisions for patients in the field. This is not something conventional cytometry traditionally worries about, but is very important for development of small, portable microfluidic devices with small-volume throughputs. It also provides a more reasonable alternative to conventional tubes of blood when sampling geriatric and newborn patients for whom a conventional peripheral blood draw can be problematical. Instead one or two drops of blood obtained by pin-prick should be able to provide statistically meaningful results for use in making real-time medical decisions without the need for blood fractionation, which is not realistic in the doctor’s office or field.

We demonstrate a multimodal imaging methodology to probe the nanoscale environment of cells. The system combines partial-wave spectroscopic (PWS) microscopy and spectroscopic photon localization microscopy (SPLM). PWS quantifies the nanoarchitecture of cells with sensitivity to structures between 20 and 200 nm. SPLM is a newly developed super-resolution imaging technique based upon the principles of single-molecule localization microscopy and spectroscopy. In addition to allowing super-resolution imaging, SPLM provides inherent molecular-specific spectroscopic information of targeted structures visualized when dyes are used. Combining both of these modalities into a single instrument allows nanoscale characterization of the super-resolution molecular imaging provided by SPLM as it relates to nanoscale structural information provided by PWS. As an example, we labeled RNA polymerase in HeLa cells and showed correlations between the locations of the RNA polymerase visualized by SPLM and the nanoscale structure of the chromatin measured by PWS. Such information is crucial in understanding the role of specific molecules in regulating the chromatin structure and gene expression. More broadly, this instrument can give insight into the molecular pathways of diseases and therapeutic treatments of those diseases, while simultaneously showing the effects on chromatin topology.

Angularly resolved elastic scattering microscopy enables the size distribution of cellular organelles to be estimated non-invasively. By comparing the angular distribution of light scattered from a cell to Mie theory models, our group is working towards obtaining quantitative estimates of mean organelle size within single cells and tracking them over time. These estimates can then be used to assess the health of the cell. We are now testing our system’s performance in measuring induced size changes in HeLa cells undergoing apoptosis, or programmed cell death. Apoptosis is induced in these cells by exposing them to a Fas ligand. Because the response of the cell occurs on the scale of a few hours, we can take multiple measurements of many cells individually before they show changes that would be visible with traditional microscopy. Additionally, we take measurements prior to exposing cells to the ligand to establish the baseline fluctuations in the organelle size estimates. Our goal is to find optical signatures that characterize the cellular progression prior to the final, most extreme stages of the apoptotic process. We would then be able to look at the behavior of multiple cells and predict when each cell will die.

A smartphone mobile medical application, previously presented as a tool for individuals with hand arthritis to assess and monitor the progress of their disease, has been modified and expanded to include extraction of anatomical features from the hand (joint/finger width, and angulation) and foot (length, width, big toe angle, and arch height index) from smartphone camera images.

Image processing algorithms and automated measurements were validated by performing tests on digital hand models, rigid plastic hand models, and real human hands and feet to determine accuracy and reproducibility compared to conventional measurement tools such as calipers, rulers, and goniometers. The mobile application was able to provide finger joint width measurements with accuracy better than 0.34 (±0.25) millimeters. Joint angulation measurement accuracy was better than 0.50 (±0.45) degrees. The automatically calculated foot length accuracy was 1.20 (±1.27) millimeters and the foot width accuracy was 1.93 (±1.92) millimeters. Hallux valgus angle (used in assessing bunions) accuracy was 1.30 (±1.29) degrees. Arch height index (AHI) measurements had an accuracy of 0.02 (±0.01).

Combined with in-app documentation of symptoms, treatment, and lifestyle factors, the anatomical feature measurements can be used by both healthcare professionals and manufacturers. Applications include: diagnosing hand osteoarthritis; providing custom finger splint measurements; providing compression glove measurements for burn and lymphedema patients; determining foot dimensions for custom shoe sizing, insoles, orthotics, or foot splints; and assessing arch height index and bunion treatment effectiveness.

Lanthanide-based upconverting nanoparticles (UCNP's) are able to efficiently convert near infrared excitation energy into visible or higher energy near infrared light, thereby presenting an attractive platform for construction of biological imaging agents. However, lack of solubility and difficulty in their surface modifications hampered development of UCNP's as general imaging agents. Previously, we have demonstrated that shape-persistent polyglutamic dendrimers, exhibiting multiple carboxylate groups as their termini, are of tight bind to UCNP surfaces, thus stabilizing them in aqueous solutions and converting them into bio-compatible imaging probes.1 However, solubility imparted by polycarboxylates was found to be negatively impacted by the presence of divalent metal cations and various macromolecules (proteins, lipids etc), which are abundant in biological systems. In this work, we report extension of our approach using Janus-type dendrimers, in which one half of the dendrimer surface features carboxylates for binding to UCNP surfaces and another is highly hydrophilic, but neutral as a result of extensive PEG-ylation. The new dendritic UCNP's proved to have superior stability and biocompatibility and allowed high-resolution imaging of brain vasculature in mice up to 1 mm deep into the cortex by means of multiphoton microscopy with continuous-wave infrared excitation sources. The synthesis of PEGylated Janus-dendrimers as well as the method of dendrimerization of UCNP's has been optimized and scaled up, thus presenting a new generally applicable methodology. References 1 Esipova, T. V.; Ye, X.; Collins, J. E.; Sakadžić, S.; Mandeville, E. T.; Murray, C. B.; Vinogradov, S. A. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 20826. Support of the NIH grant EB018464 is gratefully acknowledged.

The crucial goal of kidney-sparing surgical resection of a malignant tumor is complete removal of the cancerous tissue. The exact border between the cancerous and normal tissues is not always possible to identify by naked eye, therefore, a supplementary intraoperative diagnosis is needed. Unfortunately, intraoperative pathology methods used nowadays are time consuming and of inadequate quality rendering not definitive diagnosis. It has recently been shown that ATR-FTIR spectroscopy can be used for fast discrimination between cancerous and normal kidney tissues by analyzing the collected spectra of the tissue touch imprint smears. Most prominent differences are obtained in the wavenumber region from 950 cm^-1 to 1250 cm^-1, where the spectral bands due to the molecular vibrations of glycogen arise in the spectra of cancerous tissue smears. Such method of detection of cancerous tissue is limited by requirement to transfer the suspected tissue from the body to the FTIR instrument and stamp it on an ATR crystal of the spectrometer. We propose a spectroscopic tool which exploits the same principle of detection of cancerous cells as mentioned above, but does not require the tissue to be transferred from the body to the spectrometer. The portable spectrometer used in this design is equipped with fiber ATR probe and a sensitive liquid nitrogen cooled MCT detector. The design of the fiber probe allows the ATR tip to be changed easily in order to use only new sterilized tips for each measurement point of the tissue. It also enables sampling multiple areas of the suspected tissue with high lateral resolution which, in turn, increases accuracy with which the marginal regions between normal and cancerous tissues can be identified. Due to the loss of optical signal in the fiber probe the spectra have lower signal-to-noise ratio than in the case of standard ATR sampling setup. However, software for the spectral analysis used with the fiber probe design is still able to distinguish between cancerous and normal tissues with high accuracy.

Autofluorescence has historically been considered a nuisance in medical imaging. Many endogenous fluorophores, specifically, collagen, elastin, NADH, and FAD, are found throughout the human body. Diagnostically, these signals can be prohibitive since they can outcompete signals introduced for diagnostic purposes. Recent advances in hyperspectral imaging have allowed the acquisition of significantly more data in a shorter time period by scanning the excitation spectra of fluorophores. The reduced acquisition time and increased signal-to-noise ratio allow for separation of significantly more fluorophores than previously possible. Here, we propose to utilize excitation-scanning of autofluorescence to examine tissues and diagnose pathologies.

Spectra of autofluorescent molecules were obtained using a custom inverted microscope (TE-2000, Nikon Instruments) with a Xe arc lamp and thin film tunable filter array (VersaChrome, Semrock, Inc.) Scans utilized excitation wavelengths from 360 nm to 550 nm in 5 nm increments. The resultant spectra were used to examine hyperspectral image stacks from various collaborative studies, including an atherosclerotic rat model and a colon cancer study. Hyperspectral images were analyzed with ENVI and custom Matlab scripts including linear spectral unmixing (LSU) and principal component analysis (PCA). Initial results suggest the ability to separate the signals of endogenous fluorophores and measure the relative concentrations of fluorophores among healthy and diseased states of similar tissues. These results suggest pathology-specific changes to endogenous fluorophores can be detected using excitationscanning hyperspectral imaging. Future work will expand the library of pure molecules and will examine more defined disease states.

A non-destructive imaging technique is required for quantifying the anisotropic and heterogeneous structural arrangement of collagen in soft tissue membranes, such as bovine pericardium, which are used in the construction of bioprosthetic heart valves. Previously, our group developed a Stokes imaging polarimeter that measures the linear birefringence of samples in a transmission arrangement. With this device, linear retardance and optic axis orientation; can be estimated over a sample using simple vector algebra on Stokes vectors in the Poincaré sphere. However, this method is limited to a single path retardation of a half-wave, limiting the thickness of samples that can be imaged. The polarimeter has been extended to allow illumination of narrow bandwidth light of controllable wavelength through achromatic lenses and polarization optics. We can now take advantage of the wavelength dependence of relative retardation to remove ambiguities that arise when samples have a single path retardation of a half-wave to full-wave. This effectively doubles the imaging depth of this method. The method has been validated using films of cellulose of varied thickness, and applied to samples of bovine pericardium.

Thermal imaging based on Quadriwave Lateral Shearing Interferometry (QWLSI) is able to measure temperature fields from the temperature-induced refractive index changes of the medium surrounding a heat source. The conventional arrangement employed a continuous wave laser source to illuminate nano-particles and a wavefront analyzer (SID4Bio) to perform QWLSI. With this arrangement, a resolution of 1°C is achieved on the temperature variation measurement. We present a system implementing a synchronous detection modality. By modulating the laser source locked-in with the SID4Bio camera we get rid of the low-frequency noise and so increase the sensitivity of the temperature variation measurement.

Osteoarthritis (OA) is a very common joint disease in the aging population. Main symptom of OA is accompanied by degenerative changes of articular cartilage. Cartilage contains mostly type II collagen and proteoglycans, so it is difficult to access the quality and morphology of cartilage tissue in situ by conventional diagnostic tools (X-ray, MRI and echography) directly or indirectly. Raman spectroscopy is a label-free technique which enables to analyze molecular composition in degenerative cartilage. In this proposal, we aim to develop Raman spectroscopic system for the quality assessment of articular cartilage during arthroscopic surgery. Toward this goal, we are focusing on the proteoglycan content and collagen fiber alignment in cartilage matrix which may be associated with degenerative changes in OA, and we designed an original Raman device for remote sensing during arthroscopic surgery. In this project, we define the grading system for cartilage defect based on Raman spectroscopy, and we complete the evaluation of the Raman probing system which makes it possible to detect early stage of degenerative cartilage as a novel tool for OA diagnosis using human subject.

A fast, convenient way to determine the age of bones and ivories is important both in forensics and for classifying art objects in collections of art experts, restorers, art galleries and museums. Knowing the age of elephant tusks is also essential because there are many date-specific regulations of ivory trade. Radiocarbon dating is the standard method used to determine the age of organic materials, but it is expensive, time consuming, and damages the sample in the process. Raman spectroscopy is sensitive to rotational and vibrational molecular transitions, and also intermolecular vibrations. Therefore, it can provide information about sample make up, such as proteins and minerals, as well as detect spectral signatures associated with structural changes in molecules. Since Raman spectroscopy identifies the molecular bonds present in a sample, it is often used to determine its chemical composition. Bones and ivories contain two primary components: collagen and bioapatite. As the protein collagen deteriorates with time, its Raman signal decreases. The ratio of collagen-to-bioapatite peaks, therefore, is smaller in the older samples compared to the younger ones, providing a basis for sample dating. We employed Raman spectroscopy to non-destructively determine the age of several elephant tusk fragments. We have also used it to identify ivory imitations made of vegetable and plastic materials. Such materials have entirely different chemical composition, and their spectra are easily distinguished from those of bone and ivory. Peak fitting was employed to determine collagen and bioapatite components.

Cytokine diffusion and dynamics are central to cellular signaling, activation and migration, yet few, if any, technologies are currently able to quantify cytokine protein behavior in real-time, in a label-free fashion and, most importantly, with a spatial resolution that allows pinpointing individual cells. Photonic crystal resonant imaging offers high spatial and temporal resolution so it is an ideal platform for studying this mammalian extracellular process. Unlike other photonic biosensors, the resonant imaging technique offers very high spatial resolution (of order of a few μm), so it can be understood as an imaging array of highly sensitive biosensors. Here we propose and demonstrate a method of quantifying the secreted chemokine gradients from individual cells. Two Baby Hamster Kidney fibroblast cells lines (i.e. BHK cells) were employed in our studies, engineered to overexpress the chemokine interleukin 3 (IL-3) and the human thrombopoietin growth factor hormone (HTPO), respectively. Localized free cytokine/hormone levels were quantified permitting formation of real-time maps of localized secreted protein levels. The technology has been proven to be protein-specific, greatly minimizing non-specific binding from other carbohydrates secreted from the cells and therefore improving the signal-to-noise ratio of the maps. Ultimately, this system allows the identification of specific cells from mixed systems where diverse types of cells are present. This technique represents a powerful tool to noninvasively and nondestructively study cell behavior in-vitro, label-free, and in real-time; overcoming the inherent challenges of label-based methods.

We developed a confocal microscope and ultra-spectral light source containing Liquid Crystal Arrayed Microcavities (LCAM) as a new technology for controlled ultra-narrow optical filtering (FWHM ∼0.1 nm). LCAMs use picoliter volume Fabry–Perot-type optical cavities filled with liquid crystal for wavelength tuning. With sub-nanometer spectral resolution, we measured spectral resonance from scattering particles to validate the measurements with simulation results. We measured chicken liver and breast muscle, pig skin and muscle and compared with results from literature. The main application is the estimation of nuclear size in biological tissues for noninvasive assessment equivalent to histopathology.

This work presents a fully packaged confocal endomicroscopic system using Lissajous fiber scanner for in-vivo imaging. The confocal endomicroscopic system consists of a scanning probe part, an optical part, and an electrical part. The scanning probe uses resonant Lissajous fiber scanner based on a piezoelectric tube. The scanner successfully achieves 10 frame rate with ~ 1 kHz scanning frequencies. The probe was fully packaged for waterproofing and disinfection of medical instruments into the outer diameter of 3.4 mm. The endomicroscopic system and successfully obtained 2D reflectance imaging results, human ex-vivo imaging results and a real-time in-vivo imaging results.

Assay of cells in pleural effusion (PE) is an important means of disease diagnosis. Conventional cytology of effusion samples, however, has low sensitivity and depends heavily on the expertise of cytopathologists. We applied a polarization diffraction imaging flow cytometry method on effusion cells to investigate their features. Diffraction imaging of the PE cell samples has been performed on 6000 to 12000 cells for each effusion cell sample of three patients. After prescreening to remove images by cellular debris and aggregated non-cellular particles, the image textures were extracted with a gray level co-occurrence matrix (GLCM) algorithm. The distribution of the imaged cells in the GLCM parameters space was analyzed by a Gaussian Mixture Model (GMM) to determine the number of clusters among the effusion cells. These results yield insight on textural features of diffraction images and related cellular morphology in effusion samples and can be used toward the development of a label-free method for effusion cells assay.

Bone growth and strength is severely impacted by Hypophosphatasia (HPP). It is a genetic disease that affects the mineralization of the bone. We hypothesize that it impacts overall organization, density, and porosity of collagen fibers. Lower density of fibers and higher porosity cause less absorption and scattering of light, and therefore a different regime of transport mean free path. To find a cure for this disease, a metric for the evaluation of bone is required. Here we present an evaluation method based on our Phase Accumulation Ray Tracing (PART) method. This method uses second harmonic generation (SHG) in bone collagen fiber to model bone indices of refraction, which is used to calculate phase retardation on the propagation path of light in bone. The calculated phase is then expanded using Zernike polynomials up to 15th order, to make a quantitative analysis of tissue anomalies. Because the Zernike modes are a complete set of orthogonal polynomials, we can compare low and high order modes in HPP, compare them with healthy wild type mice, to identify the differences between their geometry and structure. Larger coefficients of low order modes show more uniform fiber density and less porosity, whereas the opposite is shown with larger coefficients of higher order modes. Our analyses show significant difference between Zernike modes in different types of bone evidenced by Principal Components Analysis (PCA).

Rod-dominated transient retinal phototropism (TRP) has been observed in freshly isolated retinas, promising a noninvasive biomarker for high resolution assessment of retinal physiology. However, in vivo mapping of TRP is challenging due to its fast time course and sub-cellular signal magnitude. By developing a line-scanning and virtually structured detection based super-resolution ophthalmoscope, we report here in vivo observation of TRP in frog retina. In vivo characterization of TRP time course and magnitude were implemented by using variable light stimulus intensities.

Because cholesterol crystals (Chcs) are a major cause of atherosclerosis, imaging Chcs in tissues with high sensitivity and specificity is important in diagnosing and predicting atherosclerosis. Polarizing microscopy (PM) has been widely used to image crystalline materials in tissues, but it has been difficult to distinguish Chcs from other crystalline materials in tissues. Thus, various methods such as fluorescent dye staining, Raman spectroscopy, and two-photon microscopy (TPM) have been developed to image Chcs with high sensitivity and specificity. However, these methods require expensive equipment or complex processes. Therefore, we have developed a low-cost, easy-to-use PM system using an LED light source that can distinguish Chcs from other crystalline materials with high sensitivity and specificity. Due to the nature of the LED spectrum in our system, collagen is displayed in yellow and Chcs in blue. In addition, we have improved the sensitivity and specificity by creating an aqueous condition on the sample. In the aqueous state, signals of yellowish collagen fibers were reduced and signals of Chcs were highlighted. The Chcs detection capability of our system was verified compared with the TPM image. In addition, clinical feasibility was shown by comparison with existing histological methods.

Hyperpolarized (HP) noble gas has attracted attention in NMR / MRI. In an ultra-low magnetic field, the effectiveness of signal enhancement by HP noble gas should be required because reduction of the signal intensity is serious. One method of generating HP noble gas is spin exchange optical pumping which uses selective excitation of electrons of alkali metal vapor and spin transfer to nuclear spin by collision to noble gas. Although SEOP does not require extreme cooling or strong magnetic field, generally it required large-scale equipment including high power light source to generate HP noble gas with high efficiency. In this study, we construct a simply generation system of HP xenon-129 by SEOP with an ultralow magnetic field (up to 1 mT) and small-scale light source (about 1W). In addition, we measure in situ NMR signal at the same time, and then examine efficient conditions for SEOP in ultra-low magnetic fields.

It is developed within a method of the active contours the approach, which is allowing to realize separation of a contour of a object of the image in case of its segmentation. This approach exceeds a parametric method on speed, but also does not concede to it on decision accuracy. The approach is offered within this operation will allow to realize allotment of a contour with high accuracy of the image and quicker than a parametric method of the active contours.

In this work we present a method to perform pituitary cell segmentation in image stacks acquired by fluorescence microscopy from pituitary slice preparations. Although there exist many procedures developed to achieve cell segmentation tasks, they are generally based on the edge detection and require high resolution images. However in the biological preparations that we worked on, the cells are not well defined as experts identify their intracellular calcium activity due to fluorescence intensity changes in different regions over time. This intensity changes were associated with time series over regions, and because they present a particular behavior they were used into a classification procedure in order to perform cell segmentation.

Two logistic regression classifiers were implemented for the time series classification task using as features the area under the curve and skewness in the first classifier and skewness and kurtosis in the second classifier. Once we have found both decision boundaries in two different feature spaces by training using 120 time series, the decision boundaries were tested over 12 image stacks through a python graphical user interface (GUI), generating binary images where white pixels correspond to cells and the black ones to background. Results show that area-skewness classifier reduces the time an expert dedicates in locating cells by up to 75% in some stacks versus a 92% for the kurtosis-skewness classifier, this evaluated on the number of regions the method found. Due to the promising results, we expect that this method will be improved adding more relevant features to the classifier.

We highlight the interest of using the Indices of Polarimetric Purity (IPPs) for the biological tissue inspection. These are three polarimetric metrics focused on the study of the depolarizing behaviour of the sample. The IPPs have been recently proposed in the literature and provide different and synthetized information than the commonly used depolarizing indices, as depolarization index (P_Δ) or depolarization power (Δ). Compared with the standard polarimetric images of biological samples, IPPs enhance the contrast between different tissues of the sample and show differences between similar tissues which are not observed using the other standard techniques. Moreover, they present further physical information related to the depolarization mechanisms inherent to different tissues. In addition, the algorithm does not require advanced calculations (as in the case of polar decompositions), being the indices of polarimetric purity fast and easy to implement. We also propose a pseudo-coloured image method which encodes the sample information as a function of the different indices weights. These images allow us to customize the visualization of samples and to highlight certain of their constitutive structures. The interest and potential of the IPP approach are experimentally illustrated throughout the manuscript by comparing polarimetric images of different ex-vivo samples obtained with standard polarimetric methods with those obtained from the IPPs analysis. Enhanced contrast and retrieval of new information are experimentally obtained from the different IPP based images.

To detect the bone quality loss in osteoporosis, we performed Raman spectroscopic analysis of sciatic nerve resection (NX) mice. Eight months after surgery, lower limbs were collected from the mice and fixed with 70% ethanol. Raman spectra of anterior cortical surface of the proximal tibia at 5 points in each bone were measured by RENISHAW inVia Raman Microscope. Excitation wave length was 785 nm. We also performed DXA and micro CT measurement to confirm the bone mineral density and bone microstructure in the osteoporotic model induced by sciatic nerve resection. In the result of Raman spectroscopy, we detected changes of Raman peak intensity ratio in carbonate/phosphate, mineral/combined proline and hydroxyproline and mineral/phenylalanine. In addition, in the result of micro CT, we found significant changes in VOX BV/TV, Trabecular number, thickness, cancellous bone mineral density, cortical thickness and cortical bone mineral density. The results suggest that not only the bone mineral density but also bone quality reduced in the NX mice. We conclude that Raman spectroscopy is a useful for bone quality assessment as a complementary technique for conventional diagnostics.

Dynamic fluorescence imaging approaches can be used to estimate the concentration of cell surface receptors in vivo. Kinetic models are used to generate the final estimation by taking the targeted imaging agent concentration as a function of time. However, tissue absorption and scattering properties cause the final readout signal to be on a different scale than the real fluorescent agent concentration. In paired-agent imaging approaches, simultaneous injection of a suitable control imaging agent with a targeted one can account for non-specific uptake and retention of the targeted agent. Additionally, the signal from the control agent can be a normalizing factor to correct for tissue optical property differences. In this study, the kinetic model used for paired-agent imaging analysis (i.e., simplified reference tissue model) is modified and tested in simulation and experimental data in a way that accounts for the scaling correction within the kinetic model fit to the data to ultimately extract an estimate of the targeted biomarker concentration.

Real-time and continuous monitoring of drug release in vivo is an important task in pharmaceutical development. Here, we devoted to explore a real-time continuous study of the pharmacokinetics of free indocyanine green (ICG) and ICG loaded in the shell-sheddable nanoparticles in tumor based on a dynamic diffuse fluorescence tomography (DFT) system: A highly-sensitive dynamic DFT system of CT-scanning mode generates informative and instantaneous sampling datasets; An analysis procedure extracts the pharmacokinetic parameters from the reconstructed time curves of the mean ICG concentration in tumor, using the Gauss-Newton scheme based on two-compartment model. Compared with the pharmacokinetic parameters of free ICG in tumor, the ICG loaded in the shell-sheddable nanoparticles shows efficient accumulation in tumor. The results demonstrate our proposed dynamic-DFT can provide an integrated and continuous view of the drug delivery of the injected agents in different formulations, which is helpful for the development of diagnosis and therapy for tumors.

The purpose of this study is to develop a estimation technique of local cell activity in cultured 3D cell aggregate with gelatin hydrogel microspheres by using Raman spectroscopy. It is an invaluable technique allowing real-time, nondestructive, and invasive measurement. Cells in body generally exist in 3D structure, which physiological cell-cell interaction enhances cell survival and biological functions. Although a 3D cell aggregate is a good model of the cells in living tissues, it was difficult to estimate their physiological conditions because there is no effective technique to make observation of intact cells in the 3D structure. In this study, cell aggregates were formed by MC3T-E1 (pre-osteoblast) cells and gelatin hydrogel microspheres. In appropriate condition MC3T-E1 cells can differentiate into osteoblast. We assume that the activity of the cell would be different according to the location in the aggregate because the cells near the surface of the aggregate have more access to oxygen and nutrient. Raman imaging technique was applied to measure 3D image of the aggregate. The concentration of the hydroxyapatite (HA) is generated by osteoblast was estimated with a strong band at 950-970 cm^-1 which assigned to PO₄^3- in HA. It reflects an activity of the specific site in the cell aggregate. The cell density in this specific site was analyzed by multivariate analysis of the 3D Raman image. Hence, the ratio between intensity and cell density in the site represents the cell activity.

Pancreatic ductal adenocarcinoma (PDAC) tumors are highly fibrotic and poorly vascularized, making systemic delivery of standard chemotherapies ineffective. Targeted nanoparticles that deliver effective doses of chemotherapeutic drugs specifically to PDACs could improve chemotherapy efficacy without the toxicities. Multiphoton microscopy, which uses ultra-short femto-second laser pulses as the excitation source, is efficient in multiphoton excitation fluorescence (MPEF) of endogenous or exogenous fluorescent macromolecular systems and the induction of highly specific second harmonic generation (SHG) signals from non-centrosymmetric macromolecules such as fibrillar collagens. Much of the traditional histological and chemical procedures associated with conventional microscopy methods, which may alter the native structure of tissue samples, can be circumvented to generate spatially and spectrally accurate 3D morphological and fine structural information. Orthotopic PDAC xenografts were established by injecting epithelioid carcinoma cell line derived from human pancreas (PANC-1 cells) into the pancreas of athymic male nude mice and were grown for 4 weeks. Near-infrared imaging agent indocyanine green (ICG) loaded calcium phosphosilicate nanoparticles (CPSNPs) including targeted CPSNPs bioconjugated with DNA Aptamer (AP1153), non-targeted CPSNPs (with no aptamer bioconjugation) and empty non-ICG containing CPSNPs were injected into the tail vein (n=4 mice/group). At 18 hours post-injection, mice were sedated and whole animal imaging was performed. To assess cellular uptake in ex vivo, whole tumor cross-sections were then imaged to spatially and spectrally localize ICG loaded nanoparticles relative to fibrotic regions (fibrillar collagen) of the tumor using MPEF and SHG imaging. Ex vivo imaging of cross-sections of whole tumors showed that cellular uptake of the targeted CPSNPs was clearly enhanced compared to untargeted CPSNPs and the 3D reconstructions showed accumulation of CPSNPs in tumor cells within the surrounding fibrotic stroma. We here conclude that multiphoton based multi-dimensional and minimally invasive imaging modalities provide spatially resolved 3D images with spectral specificities, that are sensitive enough to detect near-infrared imaging agents and to identity the bio-distributions of targeted versus untargeted drug nanoparticles in pancreatic tumor tissues.